Molecular Analysis of System N Suggests Novel Physiological Roles in Nitrogen Metabolism and Synaptic Transmission
Farrukh A. ChaudhryRichard J. ReimerDavid KrižajDiane L. BarberJon Storm‐MathisenDavid R. CopenhagenRobert H. Edwards
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The effects of cadmium (Cd2+) on Na+,K(+)-ATPase in disrupted human erythrocyte membranes and on various transmembrane Na+ and K+ transport systems in intact erythrocyte suspensions were studied. Cadmium2+ inhibited the erythrocyte Na+,K(+)-ATPase enzyme with a 50% inhibition at a Cd2+ concentration of 6.25 microM. The Cd2+ inhibition in the human erythrocyte was non-competitive with respect to Na+,K+, and ATP. Cadmium2+ exerted no acute effect, however, on the Na+,K(+)-ATPase pump activity as measured by the ouabain sensitive 86Rb uptake or Na+ efflux in intact red blood cells. Cadmium2+ also inhibited the Ca2+ dependent K+ channels in human red blood cells, whereas it had no effect on Na+,K+ cotransport, Na+,Li+ countertransport, anion carrier, and the number of active Na+ pump units. The data indicate that in human erythrocytes under acute conditions Cd2+ exerts an inhibitory effect on Na+,K(+)-ATPase enzyme in disrupted erythrocytes and the Ca2+ stimulated K+ efflux in intact red blood cells without affecting the Na+ pump, Na+,K+ cotransport, and Na+,Li+ countertransport activity.
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The worldwide dissemination of resistant bacteria has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. Modifications of membrane permeability by changing the expression of transporters alter the mechanical barrier to control the intracellular concentration of antibiotics. This first line of bacterial defense actively participates in the dissemination of multidrug-resistance phenotype. The regulation of membrane permeability and the expression of appropriate channel-forming proteins such as porins or efflux pumps is a key way controlling the intracellular concentration of β-lactams and quinolones, two prominent classes of our antibiotic arsenal. It is necessary to decipher functional, structural, and genetic aspects of the membrane transporters to understand their involvement in membrane physiology and permeability. Regarding the clinical aspect, resistant bacterial strains exhibit significant variations in transporters' expression under antibiotic pressure, demonstrating their role in the adaptation of membrane permeability. Faced with bacterial membrane adaptation, a number of scientific challenges address the drug influx and efflux in resistant isolates. By what means can we circumvent the bacterial membrane controls and bypass the barrier: by using permeabilizers and increasing the influx rate? How can we submerge the efflux activity: by synthesizing efflux blockers? Several studies carried out in these large areas have provided information for the development of a new generation of antibacterial agents exhibiting a variety of chemical–biological properties. Consequently, different chemical or natural groups of modulators of the bacterial membrane permeability have been characterized to increase the intracellular concentration of antibiotics as "escort or adjuvant" molecules. Used in combination, they can restore the activity of old antibiotics in multidrug-resistant gram-negative bacteria.
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The activity of Na+, K(+)-ATPase (ouabain-inhibited 86Rb influx), Na+, K+ cotransport (ouabain-insensitive furosemide-inhibited 86Rb or 22Na influx), Na+/Na+ exchange (ouabain-insensitive phloretin-inhibited 22Na influx) and Na+/Li+ exchange (ouabain-insensitive Na0(+)-depended Li+ efflux) as well as the passive permeability of the erythrocyte membrane for Na+, K+ and Li+ have been studied in patients with primary (microspherocytosis, hemoglobinopathy) and secondary (autoimmune) hemolytic anemia. The activities of the Na+, K(+)-pump and Na+, K(+)-cotransport were increased in patients with microspherocytosis-by 45% and 70%, respectively. In patients with hemoglobinopathy the Na+/Li+ exchange and passive permeability for K+ were increased 2-3-fold in comparison with the control with the control group. The increased passive permeability for K+ may partly be due to the increased K+, Cl(-)-cotransport. In patients with autoimmune anemia the 3-fold increase in the passive permeability for monovalent cations and the 2-fold increased activity of Na+, K+ cotransport were found. There was no significant correlation between the Na+/Na+ and Na+/Li+ exchange which suggests that the cellular mechanisms of activity control in those ion transport systems differ essentially. No correlation was found between the passive permeability for Na+ and K+ either. These data indicate that simple diffusion (leakage) is not the only pathway for the passive permeability of the erythrocyte membrane for monovalent cations.
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When the sodium pump was identified 30 years ago in Glynn's classical experiments on human red-cell Na and K fluxes (Glynn, 1956, 1957), the ouabain-insensitive monovalent cation fluxes were regarded as simply dissipative. It seemed that cell volume regulation could be explained by a two-component pump-leak model (Tosteson and Hoffman, 1960), but even then, the saturation kinetics displayed by the nonpump tracer K and Na influxes precluded a simple description as electrodiffusion. Since that time, it has become clear that ouabain-insensitive movements of Na and K occur via specific membrane transport systems, which show kinetic properties consistent with carriers or channels. A number of pathways for K transport are recognized: the Ca-activated (Gardos) channel, the NaKCl cotransport system, and the KCl cotransporter; for Na, NaKCl cotransport, Na-H and Na-Li exchanges, NaCO 3 - anion exchange via capnophorin, and the Na-dependent amino-acid transporters asc and gly are significant contributors to transport (Ellory and Tucker, 1983). It is obvious that it is necessary to characterize these transport systems in order to understand the maintenance of intracellular ionic composition and, therefore, volume regulation in red cells (as a model system) and hence other cell types.
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Abstract The identity of the genetic defect(s) in Swiss 3T3 TNR‐2 and TNR‐9 that confers nonresponsiveness to the proliferative effect of 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide‐sensitive Na + K + Cl − cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA‐nonresponsive lines TNR‐2 and TNR‐9 were determined in the presence and absence of TPA. When the rate of 86 Rb + efflux (as a tracer for K + ) was measured from each of the three cell lines, a furosemide‐ and TPA‐inhibitable component of efflux was clearly evident in parental and TNR‐9 cells but was virtually absent in TNR‐2 cells. 86 Rb + influx measurements indicated the presence in parental 3T3 cells and the TNR‐9 line of a substantial furosemide‐sensitive flux that could be inhibited by TPA. In contrast, much less furosemide‐sensitive influx was present in 3T3‐TNR‐2 cells, and it was relatively unaffected by TPA. In both parental 3T3 and 3T3‐TNR‐2 cells, most of the furosemide‐sensitive 86 Rb + influx is dependent on extracellular Na + and Cl − . The apparent affinities of the transporter for these two ions, as well as for K + , were similar in both cell lines. In parental cells, the inhibition of furosemide‐sensitive 86 Rb + influx was quite sensitive to TPA (K 1/2 ≅ 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume‐regulatory role, inhibition of Na + K + Cl − cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR‐2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3‐TNR‐2 cells (but not TNR‐9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na + K + Cl − cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of contransport activity in TNR‐2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3‐TNR‐2 cells (but not TNR‐9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na + K + Cl − contransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.
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Abstract The sodium cotransport systems comprise an important group of transport proteins which are involved in the transport of a variety of organic and inorganic solutes across the cellular membrane of animal cells. These systems play a central role in a wide variety of cellular and biochemical processes. We summarize here the current state of knowledge regarding the variety, structure and regulation of this important group of membrane proteins.
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The majority of anion transport inhibitors tend to be non-specific. This is problematic from a research point of view as caution is required when defining pathways purely based on pharmacology. Here we have tested a range of classical and putative Cl- transport inhibitors on three Cl- carrier systems (the KCl cotransporter (KCC), the NaK2Cl cotransporter (NKCC), and the Band 3 anion exchanger (AE)) found in human erythrocytes, using radiolabel tracer experiments. The study confirms the cross-reactivity of many anion transport inhibitors. However, two compounds, H25 and H156, were found to be both potent (IC50 values < 0.1 mM) and specific (at least 1000-fold more effective against one carrier compared to the other two) inhibitors of NKCC and AE, respectively.
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Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH − ) groups. The inorganic form (Hg 2+ ) is toxic to epithelial tissues and interacts with various transport proteins including the Na + pump and Cl − channels. In this study, we determined whether the Na + -K + -Cl − cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg 2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg 2+ inhibits Na + -K + -Cl − cotransport, with inhibitor constant ( K i ) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg 2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697→Leu) reveals that transmembrane domain 11 plays an essential role in Hg 2+ binding. We also show that modification of additional SH − groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.
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